Compositions and methods for treating or preventing dermal disorders

ABSTRACT

The present invention includes compositions and methods for treating or preventing certain dermal disorders including dermal atrophy, pseudoscars, actinic keratosis, seborrheic or actinic keratoses, lentigines, focal areas of dermal thickening, and coarse wrinkles. In certain embodiments, the compositions useful within the invention comprise a therapeutically effective amount of a mTORC1 inhibitor and a dermatologically acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2016/052442, filed Sep. 19, 2016, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/232,228, filed Sep. 24, 2015, allof which applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberAG039799 awarded by the National Institute of Aging/National Institutesof Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Aging of the skin is the most prominent feature of the aging process,being caused by multiple factors such as intrinsic aging process and UVlight exposure. Age-related dermal disorders include for example dermalatrophy, actinic keratosis, pseudoscars, lentigines, focal areas ofdermal thickening, and coarse wrinkles.

Dermal atrophy, also called skin atrophy or atrophy, is a disordermanifesting thinning or depression of skin due to reduction ofunderlying tissue. Dermal atrophy is a major clinical problem in theelderly population. Loss of dermal integrity leads to increasedfragility of the skin and precludes the use of intravenous lines in manycases. Impairment in wound healing is an important clinical sequelae ofreduced dermal integrity leading to an increase in the number of theinfections and complications following injury. Pseudoscars are stellatelesions that occur spontaneously in elderly individuals which can occuras senile and presenile forms. These lesions can be found in 20% ofpatients over the age of 70. Lentigines (or liver spots) are areas ofhyperpigmentation occurring with age and may represent precursor lesionsto lentigo maligna and melanoma. They may increase with age and becomecommon in middle aged and elderly individuals. Seborrheic or actinickeratosis, which comprise focal areas of epidermal thickening, canoccur, possibly representing a response to damage. Similarly, coarsewrinkles are thought to arise from a damage response. Currently,treatments for age-related dermal atrophy and related disorders includesubdermal hyaluronic acid injection, injection of botulinum toxin ortopical application of antioxidant such as vitamin C, green tea extract,and coenzyme Q, but these agents are not able to fully treat theseconditions.

Cellular senescence is a stress response activated by mammalian cellsupon exposure to several insults, such as oxidative stress, genotoxicstress, telomere attrition, or dysregulated mitogenic signaling. Thesestresses activate the senescence response by triggering two pathways:the p53/p21^(CIP1/WAF1) and the p16^(INK4A)/Rb pathway, which arerequired to establish and maintain the senescence response.Senescence-inducing stimuli can cause DNA damage and trigger a sustainedDNA damage response (DDR): in response to sustained, unresolved DNAdamage, the Ataxia Telangiectasia Mutant (ATM) kinase activates p53 andits transcriptional target p21^(CIP1/WAF1), which arrests cellularproliferation by inhibiting cell-cycle-dependent kinases. In addition,the same senescence-inducing stimuli can trigger the activation of theStress-Activated Protein Kinase p38 MAPK independently of DNA damage.p38 MAPK then can promote the arrest of the cell-cycle and establishsenescence by activating the transcription factor HBP1, which increasesthe expression of p16^(INK4A). These two pathways seem to establishsenescence with different kinetics: the DDR pathway usually mediate theinitial arrest by increasing the levels of p21^(CIP1/WAF1), and only atlater times senescence is reinforced by expression of p16^(INK4A).Furthermore, the p53 and the p38 MAPK pathways appear to be mostlyindependent of one another and are thus redundant, even thoughcross-talk between them may exist.

Mammalian/mechanistic target of rapamycin (mTOR) is an intracellularprotein complex that is responsive to both growth factors and nutrientavailability, and which also impacts mitochondrial function. It iscomprised of the TOR kinase (originally identified in yeast, and knownas mTOR in mammals), accessory proteins, and downstream mediatorsincluding the ribosomal S6 kinase (p70S6K) a key downstream target ofTOR. The TOR signaling pathway is highly conserved in eukaryotes and isfunctionally defined as the target of the highly-specific antifungal,rapamycin.

The proteins that comprise the core mTOR complex are the ser-thr kinasemTOR, also known as the FKBP-12-rapamycin associated protein (FRAP1),and mammalian lethal with SEC 13 protein 8 (mLST8). These corecomponents have the capability of forming either of two complexes,mTORC1 or mTORC2, which are distinguishable by their sensitivity torapamycin. The rapamycin-sensitive mTORC1 contains the scaffoldingprotein regulatory-associated protein of mTOR (Raptor), whereas therapamycin-insensitive complex mTORC2 contains the scaffolding proteinrapamycin-insensitive companion of mTOR (Rictor). These scaffoldingproteins function to direct mTORC1 and mTORC2 to their respectivetargets. Additional components are unique to each complex. For example,proline-rich Akt/PKB substrate 40 KDa (PRAS40) is an inhibitory proteinassociated with mTORC1, whereas the stress-activated MAPkinase-interacting protein 1 (Sin1) and the protein observed withRictor-1 (proctor) protein are associated with mTORC2. The primaryfunction attributed to the mTOR complex is the promotion of cellproliferation and growth of cells.

There is thus a need in the art for novel compositions and methods thatcan be used to treat or prevent certain age-related dermal conditions ina mammalian subject in need thereof, such as a human. The presentinvention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of treating or preventing an age-relateddermal disorder in a mammalian subject in need thereof. The inventionfurther provides a method of increasing the lifespan of a mammalianfibroblast. The invention further provides a method of preserving cellorganization of a mammalian fibroblast. The invention further provides amethod of preventing or minimizing senescence of a mammalian fibroblast.The invention further provides a kit for treating or preventing anage-related dermal disorder in a mammalian subject in need thereof.

In certain embodiments, the method comprises topically administering tothe subject a composition comprising a therapeutically effective amountof a mTORC1 inhibitor or a salt, solvate, enantiomer or diastereoisomerthereof.

In certain embodiments, the method comprises contacting the fibroblastwith a composition comprising an effective amount of a mTORC1 inhibitoror a salt, solvate, enantiomer or diastereoisomer thereof.

In certain embodiments, the age-related dermal disorder is at least oneselected form the group consisting of dermal atrophy, seborrheic oractinic keratosis, pseudoscars, lentigines, focal areas of dermalthickening, and coarse wrinkles.

In certain embodiments, the mTORC1 inhibitor is at least one selectedfrom the group consisting of BEZ235, rapamycin, everolimus, AZD8055,Temsirolimus, KU-0063794, P1-103. Torkinib, Tacrolimus, Ridaforolimus,INK-128, Voxtalisib. Torin-1, Omipalisib, OSI-027, PF-04691502,Apitolisib, GSK1059615, WYE-354, Gedatolisib, AZD-2014, Torin-2,WYE-125132, BGT226, Palomid-529, PP121, WYE-687, CH5132799, Way-600,ETP-46464, GDC-0349, XL388, and Zotarolimus, or a salt, solvate,enantiomer or diastereoisomer thereof. In other embodiments, the mTORC1inhibitor is at least one selected from the group consisting ofrapamycin, Ridaforolimus, and Everolimus, or a salt, solvate, enantiomeror diastereoisomer thereof. In yet other embodiments, the mTORC1inhibitor is rapamycin, or a salt, solvate, enantiomer ordiastereoisomer thereof.

In certain embodiments, the subject is a human. In other embodiments,the composition is applied topically to the skin of the subject.

In certain embodiments, the composition comprises about 0.001-1% byweight of the mTORC1 inhibitor, or a salt, solvate, enantiomer ordiastereoisomer thereof. In other embodiments, the mTORC1 inhibitor israpamycin, or a salt, solvate, enantiomer or diastereoisomer thereof. Inyet other embodiments, the composition further comprises adermatologically acceptable carrier. In yet other embodiments, thedermatologically acceptable carrier is at least one selected from thegroup consisting of a solvent, lubricant, emollient, emulsifier,moisturizer, thickening wax, softener, fragrance, preservative, andartificial color. In yet other embodiments, the dermatologicallyacceptable carrier comprises petrolatum.

In certain embodiments, the fibroblast is a dermal fibroblast. In otherembodiments, the dermal fibroblast is in vivo and part of a mammaliansubject's skin.

In certain embodiments, the kit comprises a composition comprising atherapeutically effective amount of a mTORC1 inhibitor, or a salt,solvate, enantiomer or diastereoisomer thereof. In other embodiments,the kit further comprises an applicator. In yet other embodiments, thekit further comprises instructions for topically administering thecomposition to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIGS. 1A-1I are a set of graphs illustrating changes in mitochondrialparameters in response to NRTIs and rapamycin. Human cardiac fibroblastswere maintained with or without rapamycin (1 nM). After 7 days exposureto NRTIs, parameters of mitochondrial status and function were examined.Grey bars represent cells maintained under standard culture conditionsand black bars represent cells maintained in the presence of rapamycin.FIG. 1A is a bar graph illustrating mitochondrial membrane potential asassessed by tetramethylrhodamine ethyl ester, perchlorate (TMRE)staining. FIG. 1B is a bar graph illustrating mitochondrial ROS levels.FIG. 1C is a bar graph illustrating mitochondrial mass. FIG. 1D is a bargraph illustrating total cellular ROS. FIG. 1E is a graph illustratingoxygen consumption/cell as a function of time following the addition ofthe mitochondrial inhibitors oligomycin, carbonyl cyanidep-triflouromethoxyphenylhydrazone (FCCP), or a combination of rotenoneand antimycin A. FIG. 1F is a bar graph illustrating the calculatedrates of basal respiration. FIG. 1G is a bar graph illustrating thecalculated rates of maximal respiration. FIG. 1H is a bar graphillustrating the calculated rates of ATP-linked respiration. FIG. 1I isa bar graph illustrating the calculated rates of proton leak. Eachmeasurement represents a minimum of triplicate cultures and allmeasurements were repeated a minimum of 2 times with similar results.Bars marked with an asterisk represent values that are significantlydifferent from relative control values at P<0.05 and bars marked with an# represent values that are significantly different betweenrapamycin-treated and untreated cells within the same treatment group(control or exposed to NRTIs).

FIGS. 2A-2H illustrate steady state levels of electron transport chainsubunits and mitochondrial proteins. FIG. 2A depicts steady state levelsof the indicated electron transport chain (ETC) proteins, along with theouter mitochondrial membrane protein voltage-dependent anion channel(VDAC), assessed by immunoblot in cardiac fibroblasts exposed to NRTIs.FIG. 2B depicts the steady state levels of Pink1, the Pink 1 cleavageproduct, and Parkin. FIG. 2C depicts the steady state levels p62 andactin. FIG. 2D depicts the results of a nanostring analysis of mRNAlevels for the ETC subunits included in NADH dehydrogenase (ubiquinone)1 beta subcomplex, 8 (NDUFB8) (complex 1). FIG. 2E depicts the resultsof a nanostring analysis of mRNA levels for ETC subunits included inubiquinol-cytochrome c reductase core protein II (UQCRC2) (complex 3).FIG. 2F depicts the results of a nanostring analysis of mRNA levels forthe ETC subunits included in succinate dehydrogenase (ubiquinone)ironsulfur subunit (SDHB) (complex 2). FIG. 2G depicts the results of ananostring analysis of mRNA levels for the ETC subunits included incytochrome c oxidase subunit I (mt-Co 1) (complex 4). FIG. 2H depictsthe results of a nanostring analysis of mRNA levels for the ETC subunitsincluded in ATP5A1 (complex 5). Each immunoblot represents a minimum oftwo independent experiments with similar results. Gray bars representdata from control cultures while black bars represent data fromrapamycin-treated cultures. Bars marked with an asterisk representvalues that are significantly different from relative control values atP<0.05 and bars marked with an # represent values that are significantlydifferent between rapamycin-treated and untreated cells within the sametreatment group (control or exposed to NRTIs). Nanostring results arerepresentative of 2 independent experiments.

FIGS. 3A-3F illustrate the finding that NRTI exposure induces thesenescence response, which is prevented by rapamycin. Human cardiacfibroblasts were maintained with or without rapamycin (1 nM) followingexposure to NRTIs for 7 days. Markers of senescence were examined. FIG.3A illustrates data from human cardiac fibroblasts showing steady statelevels of p16, p21, p53, lamin B1, and IL-6 known to be altered duringthe senescence response. Actin levels are presented as a loadingcontrol. FIG. 3B is a bar graph illustrating the percentage of cellsstaining positive for SA-β galactosidase activity. FIG. 3C is a bargraph illustrating steady state mRNA levels for p21 determined bynanostring analysis in cells exposed to NRTIs. FIG. 3D is a bar graphillustrating steady state mRNA levels for lamin B1 determined bynanostring analysis in cells exposed to NRTIs. FIG. 3E is a bar graphillustrating a comparative analysis of p21, comparing early passagecells with cells that have undergone replicative senescence as well ascells that were maintained in rapamycin containing medium allowinglifespan extension. FIG. 3F is a bar graph illustrating a comparativeanalysis of lamin B1, comparing early passage cells with cells that haveundergone replicative senescence as well as cells that were maintainedin rapamycin containing medium allowing lifespan extension. Grey barsrepresent cells maintained under standard culture conditions and blackbars represent cells maintained in the presence of rapamycin. Barsmarked with an asterisk represent values that are significantlydifferent from relative control values at P<0.05 and bars marked with an# represent values that are significantly different betweenrapamycin-treated and untreated cells within the same treatment group(e.g. control or exposed to NRTIs).

FIGS. 4A-4E illustrate the finding that ROS scavengers preventexpression of senescence markers in cells exposed to NRTIs. Humancardiac fibroblasts were treated with the mitochondrial ROS scavengermito-Q during exposure to NRTIs. FIG. 4A depicts the steady state levelsof p16, p21, p53 and lamin B1 known to be altered during the senescenceresponse. FIG. 4B is a bar graph illustrating the percentage of cellsstaining positive for lysosomal SA-β galactosidase activity. FIG. 4Cdepicts the results of an immunoblot analysis showing steady statelevels of p16, p21, p53, actin, and catalase known to be altered duringthe senescence response, after human cardiac fibroblasts were infectedwith an adenoviral vector expressing the mt-catalase protein or an emptyvector during exposure to NRTIs. Immunoblot represents a minimum of twoindependent experiments with similar results. FIG. 4D is a bar graphillustrating the percentage of cells staining positive for lysosomalSA-βgalactosidase activity, after human cardiac fibroblasts wereinfected with an adenoviral vector expressing the mt-catalase protein oran empty vector during exposure to NRTIs. FIG. 4E depicts steady statelevels of p16, p21, and lamin B1 associated with senescence. Grey barsrepresent cells maintained under standard culture conditions and blackbars represent cells maintained in the presence of rapamycin. Barsmarked with an asterisk represent values that are significantlydifferent from relative control values at P<0.05. Bars marked with an #represent values that are significantly different betweenrapamycin-treated and untreated cells within the same treatment group(control or exposed to NRTIs).

FIGS. 5A-5D illustrate phosphorylation of ribosomal S6 protein and MDM2in response to NRTI exposure. FIG. 5A depicts the phosphorylation statusof the ribosomal S6 protein and MDM2 in response to NRTI exposure asassessed by immunoblot analysis in human cardiac fibroblasts. Extractsfrom cultures maintained under standard culture conditions are shown inthe left 3 lanes while cultures maintained in the presence of 1 nMrapamycin are shown in the 2 right hand lines. Cultures were exposed to10 or 20 μM NRTIs for 7 days in the case of control cultures whilerapamycin treated cultures were exposed to 20 μM NRTIs for 7 days. FIG.5B depicts the phosphorylation status of the ribosomal S6 protein andMDM2 after cells were exposed to NRTIs for 7 days followed by incubationwith specific kinase inhibitors targeting either the p70 S6 kinase(PF-4708671) or MEK1/2 (U0126) for the final 2 hours. FIG. 5C depictsthe phosphorylation status of the ribosomal S6 protein and MDM2 aftercells were exposed to NRTIs for 7 days followed by incubation withspecific kinase inhibitors targeting MEK1/2 (U0126), Raf (GW5047), orp90RSK (BI-D1870), for the final 2 hours. An additional set of cultureswas treated with the free radical scavengers Trolox and N-acetylcysteinein combination during exposure to NRTIs. FIG. 5D depicts thephosphorylation status of the ribosomal S6 protein and MDM2 after cellswere treated with a specific p38 MAPK inhibitor (SB203580) duringexposure to NRTIs. With the exception of FIG. 5D, each immunoblotrepresents a minimum of two independent experiments with similarresults. Each immunoblot is shown with actin as a loading control.

FIGS. 6A-6G illustrate effect of Mito-Q and mt-catalase on ribosomal S6and MDM2 phosphorylation and mitochondrial activity in response toNRTIs. FIG. 6A depicts an immunoblot analysis of the phosphorylationstatus of the ribosomal S6 protein and MDM2 in cardiac fibroblastscultured in the presence of mito-Q or the inactive carrier thiaminepyrophosphate (TPP) for the duration of NRTI exposure. FIG. 6B depictsan immunoblot analysis for phosphorylation of the ribosomal S6 proteinand MDM2 in cardiac fibroblasts infected with adenoviral particlesharboring a construct expressing the mt-catalase or an empty viralvector prior to NRTI exposure. Each immunoblot represents a minimum oftwo independent experiments with similar results and is shown with actinas a loading control. FIG. 6C is a Seahorse Bioanalyzer analysisillustrating calculated rates of basal respiration after cardiacfibroblasts treated with mito-Q or TPP during exposure to NRTIs. FIG. 6Dis a Seahorse Bioanalyzer analysis illustrating calculated rates ofmaximal respirations after cardiac fibroblasts treated with mito-Q orTPP during exposure to NRTIs. FIG. 6E is a Seahorse Bioanalyzer analysisillustrating calculated rates of ATP-linked respiration after cardiacfibroblasts treated with mito-Q or TPP during exposure to NRTIs. FIG. 6Fis a Seahorse Bioanalyzer analysis illustrating calculated rates ofproton leak after cardiac fibroblasts treated with mito-Q or TPP duringexposure to NRTIs. FIG. 6G is a bar graph illustrating the meanfluorescence intensity of mitochondrial ROS. Bars with an asteriskrepresent values that are significantly different from relative controlvalues at P<0.05 and bars marked with an # represent values that aresignificantly different between TPP and mito-Q-treated cells within thesame treatment group (control or exposed to NRTIs).

FIGS. 7A-7B illustrate phosphorylation status of the ribosomal S6protein and MDM2 in response to oxidative stress. FIG. 7A depictsphosphorylation status of the ribosomal S6 protein and MDM2 afterserum-deprived cardiac fibroblasts exposed to increasing concentrationsof hydrogen peroxide for 2 hours. Serum-stimulated cultures are includedin the right hand lane as a positive control for growth factorstimulation of ribosomal S6 phosphorylation. FIG. 7B depicts thephosphorylation status of the ribosomal S6 protein in cells exposed tothe indicated concentrations of rotenone for 16 hours. Serum deprivedcardiac fibroblasts were exposed to rotenone at concentrations rangingfrom 1 to 50 nM. Steady state levels of actin are presented as a controlfor equal protein loading.

FIGS. 8A-8C illustrates localization of Raptor to mitochondria in thepresence of NRTIs. FIG. 8A is a set of images illustrating cellsinfected with an adenoviral construct harboring an expression constructthat produces a GFP protein fused to a mitochondrial targeting sequence(green). These cells were fixed and stained with an antibody thatrecognizes Raptor (red) and counter stained with4′,6-diamidino-2-phenylindole (DAPI) to visualize DNA (blue). FIG. 8B isa representative confocal image of control fibroblasts (not exposed toNRTIs) illustrating a co-localization event in fibroblasts expressingthe mitochondrial GFP following exposure to NRTIs for 7 days. FIG. 8C isa bar graph illustrating the quantification of co-localization events asdetermined by both confocal and deconvolution microscopy. Bars markedwith an asterisk represent values that are significantly different fromrelative control values at P<0.05 and bars marked with an # representvalues that are significantly different between rapamycin-treated anduntreated cells within the same treatment group (control or exposed toNRTIs). Co-localization experiments were performed by four independentevaluators over a series of experiments examining Raptor and mt-GFPco-localization by both confocal microscopy and deconvolutionmicroscopy. Quantitative data were generated from counts usingdeconvolution microscopy.

FIGS. 9A-9I illustrates the finding that fibroblasts display senescenceelevated mitochondrial ROS and enhanced phosphorylation of ribosomal S6protein and MDM2. FIG. 9A is a bar graph illustrating the levels ofmitochondrial ROS in early passage and senescent cardiac fibroblasts.FIG. 9B is a bar graph illustrating the levels of total cellular ROS inearly passage and senescent cardiac fibroblasts. Grey bars representcells maintained under standard culture conditions and black barsrepresent cells maintained in the presence of 1 nM rapamycin. FIG. 9C isa graph illustrating oxygen consumption rate normalized to cell number,as a function of time following the addition of the mitochondrialinhibitors oligomycin. FCCP, or a combination of rotenone and antimycinA. FIG. 9D is a Seahorse Bioanalyzer analysis illustrating basalrespiration of mitochondrial function in human cardiac fibroblasts. FIG.9E is a Seahorse Bioanalyzer analysis illustrating maximal respirationof mitochondrial function in human cardiac fibroblasts. FIG. 9F is aSeahorse Bioanalyzer analysis illustrating ATP-linked respiration ofmitochondrial function in human cardiac fibroblasts. FIG. 9G is aSeahorse Bioanalyzer analysis illustrating proton leak of mitochondrialfunction in human cardiac fibroblasts. FIG. 9H depicts protein lysatesderived from cells at increasing population doublings probed for markersof senescence, p21 and p16, as well as the phosphorylated forms of theribosomal S6 and MDM2 proteins. FIG. 9I depicts protein lysates probedfor the phosphorylation status of the ribosomal S6 protein and MDM2,after fully senescent cultures were treated with the ROS scavengersTrolox and N-acetylcysteine, the p90RSK inhibitor BI-D1870, or infectedwith adenoviral particles harboring a construct that expresses themt-catalase protein or an empty vector control. Bars marked with anasterisk represent values that are significantly different from relativecontrol values at P<0.05, and bars marked with an # represent valuesthat are same treatment group (control or exposed to NRTIs).

FIG. 10 is a schematic illustration of a model for mTORC1 integration ofmultiple signals to generate growth response or senescence arrest. Asdemonstrated herein, additional inputs to mTORC1 exist in the form ofcellular redox status and mitochondrial function that may redirectmTORC1 to support a senescent growth arrest through the p70 S6 kinasemediated modulation of MDM2 and p53 activity. These connections areshown in red (mTORC1→p7056K-|MDM2-|p53→stabilization→p53→senescence).

FIGS. 11A-11F illustrate status of p53 in human cardiac fibroblastcultures exposed to NRTIs and in rapamycin-treated cultures. FIG. 11A isa bar graph illustrating comet assay results for cells exposed to NRTIsor hydrogen peroxide as a positive control. The white bar representscontrol data; grey bar represents data from cells exposed to 10 or 20μg/ml NRTI for 7 days; and the black bar represents data from cellsexposed to 200 μM hydrogen peroxide for 2 hours. An asterisk representsvalues that are significantly different from relative control values atP<0.05. FIG. 11B depicts the results of an immunoprecipitationexperiment using antibodies against p53. Cell lysates from control andrapamycin-treated cultures, with or without NRTI exposure for 7 days,were subjected to immunoprecipitation using anti-p53 antibodies followedby immunoblot analysis for MDM2 and subsequently for p53. FIG. 11Cdepicts levels of p53, MDM2, and phosphorylated MDM2 in the samples usedfor immunoprecipitation in FIG. 11A. FIG. 11D depicts levels of p53after control and rapamycin-treated cultures were exposed to NRTIs for 7days followed by a 2-hour incubation with MG132 to inhibit proteasomeactivity. Modified p53 refers to higher molecular weight formsrecognized by the anti-p53 antibody. FIG. 11E is an image illustratingcytosolic level of MDM2 without exposure to NRTIs, and an imageillustrating cytosolic level of MDM2 with exposure to NRTIs.Representative photomicrographs of cells stained for MDM2 (red) andcounter stained for DNA (blue). FIG. 11F is a bar graph illustratingrelative intensity of cytosolic MDM2 staining as determined by Image JAnalysis.

FIGS. 12A-12D are a set of images illustrating mitochondrial associationof Raptor. FIGS. 12A-12B depict representative photomicrographs of cellsexpressing the mt-GFP protein (green) stained for Raptor (red) undercontrol conditions. FIGS. 12C-12D depict representative photomicrographsof cells expressing the mt-GFP protein stained for Raptor followingexposure to NRTIs. Co-localization events are indicated by arrows.

FIG. 13 is a graph illustrating the finding that rapamycin treatmentprovides lifespan extension in human fibroblasts. Human fibroblast cellswere growth in culture medium with or without rapamycin (1 nM). Cultureswere split every 7 days and reseeded at identical cell number/cm² eachweek. The lifespan of normal human fibroblasts is counted by the numberof times that the cells double. Rapamycin treated grow well beyond thenormal lifespan for these cells.

FIG. 14 illustrates the finding that rapamycin preserves cellorganization during aging of human fibroblasts. Human fibroblastsmaintained in the presence of low doses of rapamycin maintained anorderly growth pattern while untreated fibroblasts lost their ability toproperly orient themselves with age. The ability to organize is acritical element of normal fibroblast function and contributes to tissueintegrity in normal tissue. The disorganization which occurs with agecontributes to functional decline.

FIG. 15 are a set of images illustrating the finding that rapamycinpreserves mitochondrial network in the face of damage. Panel A depictsthat human fibroblasts expressing a fluorescent protein in themitochondrial display a green mitochondrial network. Panel B depictsthat rapamycin treated cells have a normal mitochondrial network. PanelC depicts that the mitochondrial network is destroyed by exposure to amitochondrial toxin, ethidium bromide (EthBr). Panel D depicts thatrapamycin treated cells are able to maintain their mitochondrial networkfollowing exposure to EthBr.

FIG. 16 is a bar graph illustrating the finding that rapamycin increasesthe survival of human fibroblasts following mitochondrial stress. Humanfibroblasts were exposed to ethidium bromide for 7 days and cellviability measured at that time.

FIG. 17 is a bar graph illustrating the finding that rapamycin preventsthe expression of genes that cause senescence. The p21 gene makes aprotein that is critical for senescence of human fibroblasts. Cellsgrown in the presence of rapamycin do not produce p21 as they age whilethe control cells express p21 and enter senescence.

FIG. 18 is a bar graph illustrating the finding that rapamycin preventsthe expression of genes related to inflammation. The IL-6 gene makes aprotein that activates the immune system to recruit immune cells into anarea of tissue damage. Cells grown in the presence of rapamycin do notproduce IL-6 as they age while the control cells express IL-6 and entersenescence.

FIG. 19 is a bar graph illustrating the finding that rapamycin increasesdermal thickness in atrophic skin. Dermal thickness was measured using aMitoutoyo digital caliper with certified accuracy to 0.001 mm following14 days of rapamycin treatment in an emulsified gel preparation.

FIG. 20 is a photograph of a seborrheic keratosis lesion before andafter 21 days of treatment with 10 μM rapamycin in an emulsified gelpreparation as in FIG. 19. A significant reduction in severity of thelesion was apparent upon visual inspection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to the unexpecteddiscovery that the compositions and methods of the invention can be usedto treat or prevent age-related dermal disorders including, but notlimited to, dermal atrophy, seborrheic keratosis, actinic keratosis,pseudoscars, lentigines, focal areas of dermal thickening, and coarsewrinkles. In certain embodiments, the compositions and methods of theinvention are useful for treating or preventing dermal atrophy in asubject in need thereof. In other embodiments, the compositions of theinvention comprises therapeutically effective amounts of at least onemTORC1 inhibitor. In yet other embodiments, the compositions of theinvention comprise the mTORC1 inhibitor as the only ingredient that isactive against the age-related dermal condition. In yet otherembodiments, the mTORC1 inhibitor is also a mTORC2 inhibitor.

The compositions and methods of the invention enhance lifespan offibroblasts and improve the growth and stress resistance of normalfibroblasts. Without wishing to be limited by any theory, this may beassociated with a decrease in inflammatory cytokine production. Incertain embodiments, delivery of therapeutically effective amounts of amTORC1 inhibitor to the dermal layers induces mesenchymal responses thatinfluence dermal homeostasis. In other embodiments, delivery oftherapeutically effective amounts of a mTORC1 inhibitor to the dermallayers produces an increase in dermal thickness and improvement in skinfunction.

As demonstrated herein, mitochondrial ROS was identified as a novelinput for mTORC1. Based upon both fluorescent indicators and the impactof ROS scavengers, the present results support the finding thatmitochondrial-generated ROS serves to activate mTORC1 (FIG. 10). This isreflected by increased phosphorylation of both the ribosomal S6 proteinand MDM2 observed in the experimental setting. Inhibition of mTORC1 byrapamycin prevented these responses, as did interventions aimed atreducing mitochondrial ROS, such as the expression of amitochondrial-targeted catalase or treatment with ROS scavengers.Analysis of oxygen consumption as a measure of mitochondrial activityrevealed an increase in basal and ATP-linked respiration in bothsettings, cells exposed to NRTIs as well as in those in replicativesenescence. Similarly, in both settings proton leak and mitochondrialROS production were increased. These findings suggest that thegeneration of mitochondrial ROS serves as a trigger for the activationof mTORC1. This interpretation is supported by the fact thatinterventions designed to reduce mitochondrial ROS reducedphosphorylation of the ribosomal S6 protein.

In terms of downstream consequences of mitochondrial ROS induction ofmTORC1/p70S6K activity, MDM2 phosphorylation appeared to lead to astabilization of p53 and increased expression of downstream targets suchas p21. Consistent with this, decreased association of p53 with MDM2 wasshown by co-immunoprecipitation and increased cytosolic MDM2 in cellsexposed to NRTIs. In addition, it was observed a decrease in highmolecular weight forms of p53 in cells exposed to NRTIs, whilerapamycin-treated cells contained elevated levels of these highmolecular weight forms of p53 that were visible only followinginhibition of the proteasome. This effect of rapamycin on p53 mayunderlay the lack of activation of p53 target genes, such as p21, inrapamycin-treated cells and contributed to the delayed senescenceobserved when cells were cultured in the presence of rapamycin atconcentrations sufficient to influence mTORC1 signaling but notsufficient to completely block proliferation.

In certain aspects, the present results show that mTORC1 signalingthrough the p70S6K may be responsive to ROS generated by mitochondria.Activation of mTORC1/p70S6K occurred in settings of mitochondrialdysfunction, replicative senescence, and in aged tissue. Rapamycinameliorated both the mitochondrial ROS production and blocks themTORC1/p70S6K response. These effects of rapamycin supported thebeneficial effects observed in terms of longevity and in age-relateddisorders like dermal atrophy following rapamycin treatment.

Definitions

As used herein, each of the following terms have the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics and chemistry are those well-known andcommonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount a temporal duration, and the like, the term “about” ismeant to encompass variations of ±20% or ±10%, ±5%, ±1%, or ±0.1% fromthe specified value, as such variations are appropriate to perform thedisclosed methods.

As used herein, “dermatologically acceptable carrier” or“dermatologically acceptable excipient” refers to the compositions orcomponents that are suitable for use in contact with human keratinoustissue without undue toxicity, incompatibility, instability, allergicresponse, and the like.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated, theanimal's health continues to deteriorate. A “disorder” in an animal is astate of health in which the animal is able to maintain homeostasis, butin which the animal's state of health is less favorable than it would bein the absence of the disorder. Left untreated, a disorder does notnecessarily cause a further decrease in the animal's state of health.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” or “pharmaceutically effective amount” of a compoundare used interchangeably to refer to the amount of the compoundsufficient to provide a beneficial effect to the subject to which thecompound is administered. The term to “treat,” as used herein, meansreducing the frequency with which symptoms are experienced by a patientor subject or administering an agent or compound to reduce the severitywith which symptoms are experienced. An appropriate therapeutic amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “FTC” refers to emtricitabine or a salt orsolvate thereof.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression that can be usedto communicate the usefulness of a compound, composition, assay ormethod of the invention in a kit for suppressing or reducing systemicimmune response in a subject. The instructional material of the kit ofthe invention can, for example, be affixed to a container which containsthe identified compound, composition, assay, or methods of the inventionor be shipped together with a container that contains the identifiedcompound, composition, assay, or method. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound,composition, assay, or method be used cooperatively by the recipient.

As used herein, the term “modulate” means, with respect to diseasestates or conditions associated with binding of a compound of thepresent invention to a receptor contemplated in the present invention,to produce, either directly or indirectly, an improvement or lesseningof a condition or disease state which was, prior to administration of acompound according to the present invention, sub-optimal and in manycases, debilitating and even life threatening. Modulation may occur byvirtue of agonist activity, antagonist activity or mixedagonist/antagonist activity (depending on the receptor site).

As used herein, the term “NRTI” refers to a nucleotide/nucleosidereverse transcriptase inhibitor.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the composition, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutical composition” or “composition”refers to a mixture of at least one compound useful within the inventionwith other chemical components, such as carriers, stabilizers, diluents,dispersing agents, suspending agents, thickening agents, and/orexcipients. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to: intravenous,oral, aerosol, parenteral, ophthalmic, pulmonary, intracranial andtopical administration. In certain embodiments, the administrationcomprises topical administration.

As used herein, a “subject” refers to a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. In certainembodiments, the subject is human.

As used herein, the term “TDF” refers to tenofovir disoproxil fumarate,or a salt or solvate thereof.

As used herein, “topical administration” or “topical application” refersto a medication applied to body surfaces such as the skin or mucousmembranes.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., acomposition useful within the invention (alone or in combination withanother pharmaceutical agent), to a subject, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a subject (e.g., for diagnosis or ex vivo applications), who has adisease or disorder, a symptom of a disease or disorder or the potentialto develop a disease or disorder, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease or disorder, the symptoms of the disease or disorder or thepotential to develop the disease or disorder. Such treatments may bespecifically tailored or modified, based on knowledge obtained from thefield of pharmacogenomics.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Compositions

The composition of the invention comprises a therapeutically effectiveamount of a mTORC1 inhibitor, or salt, solvate, enantiomer ordiastereoisomer thereof. In certain embodiments, the mTORC1 inhibitor isat least one selected from the group consisting of BEZ235, rapamycin,everolimus, AZD8055, Temsirolimus, KU-0063794, PI-103, Torkinib,Tacrolimus, Ridaforolimus, INK-128. Voxtalisib, Torin-1, Omipalisib,OSI-027, PF-04691502, Apitolisib, GSK1059615, WYE-354, Gedatolisib,AZD-2014, Torin-2, WYE-125132, BGT226, Palomid-529, PP121, WYE-687,CH5132799, Way-600, ETP-46464, GDC-0349. XL388, and Zotarolimus.

BEZ235 is also known as2-methyl-2-(4-(3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl)phenyl)propanenitrile,and has a formula of:

Rapamycin is also known as (3S,6R,7E,9R,1R,12R,14S,15E,17E,19E,21 S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4-oxaazacyclohentriaconine-1,5,11,28,29(4H,6H,31H)-pentone, and has a formula of:

Everolimus is also known asdihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone, and has aformula of:

AZD8055 is also known as5-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol,and has a formula of:

Temsirolimus is also known as 42-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin, and has a formula of:

PI-103 is also known as3-[4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]-phenol,and has a formula of:

KU-0063794 is also known as(5-(2-((2R,6S)-2,6-dimethylmorpholino)-4-morpholinopyrido[2,3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol,and has a formula of:

Torkinib is also known as2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol,and has a formula of:

Tacrolimus is also known as 3S-13R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone,and has a formula of:

Ridaforolimus is also known as 42-(dimethylphosphinate)-rapamycin, andhas a formula of:

INK-128 is also known as3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine,and has a formula of:

Voxtalisib is also known asN-[4-[[[3-[(3,5-dimethoxyphenyl)amino]-2-quinoxalinyl]amino]sulfonyl]phenyl]-3-methoxy-4-methyl-benzamide,and has a formula of:

Torin-1 is also known as1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one, and has aformula of:

Omipalisib is also known as2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl)benzenesulfonamide, and has a formula of:

OSI-027 is also known as (1r,4r)-4-(4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-7-yl)cyclohexane-1-carboxylic acid, and hasa formula of:

PF-04691502 is also known as2-amino-8-((1r,4r)-4-(2-hydroxyethoxy)cyclohexyl)-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-oneand has a formula of:

Apitolisib is also known as(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-v1)-2-hydroxypropan-1-one, and has a formula of:

GSK1059615 is also known as(Z)-5-((4-(pyridin-4-yl)quinolin-6-yl)methylene) thiazolidine-2,4-dione,and has a formula of:

WYE-354 is also known as4-[6-[4-[(methoxycarbonyl)amino]phenyl]-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinecarboxylicacid methyl ester, and has a formula of:

Gedatolisib is also known as1-(4-(4-(dimethylamino)piperidine-1-carbonyl)phenyl)-3-(4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)urea,and has a formula of:

AZD-2014 is also known as3-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N-methylbenzamide,and has a formula of:

Torin-2 is also known as9-(6-amino-3-pyridinyl)-1-[3-(trifluoromethyl)phenyl]-benzo[h]-1,6-naphthyridin-2(1H)-one,and has a formula of:

WYE-125132 is also known asN-[4-[1-(1,4-dioxaspiro[4.5]dec-8-yl)-4-(8-oxa-3-azabicyclo[3.2.1]oct-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl]phenyl]-N′-methyl-urea,and has a formula of:

BGT226 is also known as8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one,and has a formula of:

Palomid-529 is also known as3-(4-methoxybenzyloxy)-8-(1-hydroxyethyl)-2-methoxy-6H-benzo[c]chromen-6-one,and has a formula of:

PP121 is also known as1-cyclopentyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,and has a formula of:

WYE-687 is also known as methyl4-(4-morpholino-1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenylcarbamate, andhas a formula of:

CH5132799 is also known as5-(7-(methylsulfonyl)-2-morpholino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrimidin-2-amine,and has a formula of:

WAY-600 is also known as6-(1H-indol-5-yl)-4-morpholino-1-(1-(pyridin-3-ylmeth)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidine,and has a formula of:

ETP-46464 is also known asα,α-dimethyl-4-[2-oxo-9-(3-quinolinyl)-2H-[1,3]oxazino[5,4-c]quinolin-1(4H)-yl]-benzeneacetonitrile,and has a formula of:

GDC-0349 is also known asN-ethyl-N′-[4-[5,6,7,8-tetrahydro-4-[(3S)-3-methyl-4-morpholinyl]-7-(3-oxetanyl)pyrido[3,4-d]pyrimidin-2-yl]phenyl]-urea,and has a formula of:

XL388 is also known as[7-(6-amino-3-pyridinyl)-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl][3-fluoro-2-methyl-4-(methylsulfonyl)phenyl]-methanone,and has a formula of:

Zotarolimus is also known as42-deoxy-42-(1H-tetrazol-1-yl)-(42S)-rapamycin, and has a formula of:

In certain embodiments, the mTORC1 inhibitor contemplated in theinvention is rapamycin. In other embodiments, the mTORC1 inhibitor maybe a modified form of rapamycin with improved delivery to specificintracellular compartments or organelles, such as the mitochondria, thenucleus, the lysosome, and/or the endoplasmic reticulum.

In certain embodiments, the therapeutically effective amount of a mTORC1inhibitor in the composition ranges from about 0.001% to about 1% byweight. In other embodiments, the therapeutically effective amount byweight of the mTORC1 inhibitor in the composition ranges from about0.002% to about 0.75%, about 0.005% to about 0.5%, about 0.008% to about0.25%, about 0.01% to about 0.2%, about 0.02% to about 0.15%, or about0.03% to about 0.1%.

In certain embodiments, the composition of the invention furthercomprises a dermatologically acceptable carrier. The compositions of thepresent invention may comprise from about 60% to about 99.9%,alternatively from about 70% to about 95%, and alternatively from about80% to about 90%, of a dermatologically acceptable carrier. In certainembodiments, the dermatologically acceptable carrier is at leastselected from the group consisting of solvent, lubricant, emollient,emulsifier, moisturizer, thickening wax, softener, fragrance,preservative, and artificial color(s). In other embodiments, thedermatologically acceptable carrier is at least one selected from thegroup consisting of water, fatty alcohols, and volatile organicalcohols. One non-limiting example of the dermatologically acceptablecarrier is petrolatum.

Methods

In one aspect, the invention provides methods of increasing the lifespanof mammalian fibroblasts. In another aspect, the invention providesmethods of preserving cell organization in mammalian fibroblasts. In yetanother aspect, the invention provides methods of preventing orminimizing senescence in mammalian fibroblasts. In yet another aspect,the invention provides methods of treating or preventing age-relateddermal disorders including dermal atrophy, pseudoscars, seborrheic oractinic keratosis, lentigines, focal areas of dermal thickening, andcoarse wrinkles in a mammalian subject.

In certain embodiments, the methods of the invention comprise topicallyadministering to the subject a therapeutically effective amount of amTORC1 inhibitor, which is optionally formulated in a dermallyacceptable composition. In other embodiments, the compositions of theinvention comprises a therapeutically effective amount of a mTORC1inhibitor. In yet other embodiments, the composition further comprises adermatologically acceptable carrier. In yet other embodiments, thecomposition is applied topically to the affected skin area of thesubject.

In certain embodiments, topical formulations of the compositionscontemplated within the invention are used for treating dermal atrophy.In other embodiments, the invention provides a topical cream comprisinga therapeutically effective amount of rapamycin for treating orpreventing dermal atrophy.

In certain embodiments, dermal atrophy is evaluated by measurement ofthe dermal layer utilizing a calibrated digital caliper measurement ofthe dermal layer. In other embodiments, improvement in seborrheickeratosis, lentigines, pseudoscars, coarse wrinkles, and epidermalthickening is evaluated through an investigator evaluation rating scaleof 1-4, in which 1 is normal with no sign of lesion; 2 represents minorlesions; 3 represents lesions that are distinct features relative tonormal skin; and 4 represents lesions that are of high severity. In yetother embodiments, lesions can be examined visually or with the aid ofimage analysis software such as ImageJ, an open source image analysissoftware available from the National Institutes of Health. In yet otherembodiments, lesions are evaluated by area measurement using manualmeasurement of the lesion or through analysis of images taken byinvestigators or research study staff.

Formulations

The relative amounts of the active ingredient, the dermatologicallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated. By way of example, thecomposition may comprise between about 0.001% and about 1% (w/w) of amTORC1 inhibitor. In other embodiments, the therapeutically effectiveamount by weight of the mTORC1 inhibitor in the composition ranges fromabout 0.002% to about 0.75%, about 0.005% to about 0.5%, about 0.008% toabout 0.25%, about 0.01% to about 0.2%, about 0.02% to about 0.15%, orabout 0.03% to about 0.1%.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

The composition of the invention can be administered to a mammal asfrequently as several times daily, or it may be administered lessfrequently, such as once a day, once a week, once every two weeks, oncea month, or even less frequently, such as once every several months oreven once a year or less.

Dosing regimens for administering the compositions of the invention maybe once a day or twice a day. The frequency of the application and theconcentration of the active agent is dependent on the skin condition andthe response of the dermis. Application can be continued to achieve thedesired effect on the dermis and the frequency of application can bereduced after a satisfactory result has been obtained. In certainembodiments, the administration lasts a minimum of 2 weeks to achieveresults. Applications can continue beyond the initial 2 week period toobtain continued improvement and the frequency of application can bereduced once this result has been achieved. Applications may continueover the course of years with variable levels of application based uponthe relative severity of lesions at any one time.

It is understood that the amount of the composition of the inventiondosed per day may be administered, in non-limiting examples, every day,every other day, every 2 days, every 3 days, every 4 days, or every 5days. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, and so forth.

In certain embodiments, the compositions of the invention are formulatedusing one or more dermatologically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a mTORC1 inhibitor and adermatologically acceptable carrier. Dermatologically acceptablecarriers, which are useful, include, but are not limited to, glycerol,water, saline, ethanol and other dermatologically acceptable saltsolutions such as phosphates and salts of organic acids. Examples ofthese and other dermatologically acceptable carriers are described inRemington's Pharmaceutical Sciences (1991. Mack Publication Co., NewJersey).

The compositions of the present invention may comprise from about 60% toabout 99.9%, alternatively from about 70% to about 95%, andalternatively from about 80% to about 90%, of a dermatologicallyacceptable carrier. In certain embodiments, the dermatologicallyacceptable carrier is at least selected from the group consisting ofsolvent, lubricant, emollient, emulsifier, moisturizer, thickening wax,softener, fragrance, preservative, and artificial color(s). In otherembodiments, the dermatologically acceptable carrier is at least oneselected from the group consisting of water, fatty alcohols, andvolatile organic alcohols. One non-limiting example of thedermatologically acceptable carrier is petrolatum.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionsmay be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate or gelatin.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for topical administration, known to the art. Thepharmaceutical preparations may be sterilized and if desired mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure buffers,coloring, flavoring and/or aromatic substances and the like. They mayalso be combined where desired with other active agents, e.g., otheranalgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; coloring agents;preservatives; physiologically degradable compositions such as gelatin;aqueous vehicles and solvents; oily vehicles and solvents; suspendingagents; dispersing or wetting agents; emulsifying agents, demulcents;buffers; salts; thickening agents; fillers; emulsifying agents;antioxidants; antibiotics; antifungal agents; stabilizing agents; andpharmaceutically acceptable polymeric or hydrophobic materials. Other“additional ingredients” that may be included in the pharmaceuticalcompositions of the invention are known in the art and described, forexample in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa.), which is incorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an antioxidant and a chelating.Preferred antioxidants for some compounds are BHT, BHA, α-tocopherol andascorbic acid in the preferred range of about 0.01% to 0.3% and morepreferably BHT in the range of 0.03% to 0.1% by weight by total weightof the composition. Preferably, the chelating agent is present in anamount of from 0.01% to 0.5% by weight by total weight of thecomposition. Particularly preferred chelating agents includeaminopolycarboxylic acid salts (e.g. disodium ethylenediaminetetraaceticacid) and citric acid in the weight range of about 0.01% to 0.20% andmore preferably in the range of 0.02% to 0.10% by weight by total weightof the composition. The chelating agent is useful for chelating metalions in the composition which may be detrimental to the shelf life ofthe formulation.

Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Such formulations maybe applied to the skin directly or through the use of swabs,applicators, spatulas and the like, as well as in the form oftransdermal patches. In certain embodiments, the patch minimizes loss ofpharmaceuticals through washing, friction, scratching and/or rubbing ofthe skin. In other embodiments, the patch increases absorption of thepharmaceutical through the skin, while minimizing the exposure of theskin to the pharmaceutical.

Topically administrable formulations contemplated within the inventionmay, for example, comprise from about 0.001% to about 1% (w/w) a mTORC1inhibitor, although the concentration of the mTORC1 inhibitor may be ashigh as the solubility limit of the active ingredient in the solvent.Formulations for topical administration may further comprise one or moreof the additional ingredients described herein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, U.S. Pat. No.6,323,219).

In alternative embodiments, the topical formulation further comprisesother ingredients such as adjuvants, anti-oxidants, chelating agents,surfactants, foaming agents, wetting agents, emulsifying agents,viscosifiers, buffering agents, preservatives, and the like. In otherembodiments, a permeation or penetration enhancer is included in theformulation and is effective in improving the percutaneous penetrationof the active ingredient into and through the stratum corneum withrespect to a composition lacking the permeation enhancer. Variouspermeation enhancers, including oleic acid, oleyl alcohol,ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide,polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill inthe art. In another aspect, the topical formulation may further comprisea hydrotropic agent, which functions to increase disorder in thestructure of the stratum corneum, and thus allows increased transportacross the stratum corneum. Various hydrotropic agents such as isopropylalcohol, propylene glycol, or sodium xylene sulfonate, are known tothose of skill in the art.

Additional non-active ingredients in the topical formulation are wellknown in the art. These ingredients include, but are not limited to,humectants, emollients, pH stabilizing agents, chelating agents, gellingagents, thickening agents, emulsifiers, binders, buffers, carriers,anti-oxidants, etc. Additional examples of such ingredients are includedin the U.S. Food & Drug Administration, Inactive Ingredients forApproved Drugs, available online. Addition discussion and potentialnon-active ingredients that may be included in formulations can be foundin “The Science and Practice of Pharmacy”, 21st Edition. LippincottWilliams & Wilkins, Philadelphia, Pa. (2006).

In certain embodiments, a gel formulation of the invention comprisesabout 0.001% to about 1% (w/w) of a mTORC1 inhibitor, about 20-50% (w/w)dimethyl sulfoxide (DMSO), about 10-20% (w/w) polypropylene glycol,about 10-40% (w/w) polyethylene glycol (PEG) with a molecular weightfrom 100-800 (PEG100-PEG800), about 1-2% (w/w) gelling agents, and about0-50% Water.

In other embodiments, a gel formulation of the invention comprises about0.001% to about 1% (w/w) of rapamycin, about 20-50% (w/w) dimethylsulfoxide (DMSO), about 10-20% (w/w) polypropylene glycol, about 10-40%(w/w) polyethylene glycol (PEG) with a molecular weight from 100-800(PEG100-PEG800), about 1-2% (w/w) gelling agents, and about 0-50% Water.

In yet other embodiments, a solution or spray formulation of theinvention comprises about 0.001% to about 1% (w/w) of a mTORC1 inhibitorin an aqueous solution having about 10-50% (w/w) of DMSO and about10-50% (w/w) of PEG.

In yet other embodiments, a solution or spray formulation of theinvention comprises about 0.001% to about 1% (w/w) of rapamycin in anaqueous solution having about 10-50% (w/w) of DMSO and about 10-50%(w/w) of PEG.

In yet other embodiments, a cream or lotion formulation of the inventioncomprises about 0.001% to about 1% (w/w) of a mTORC1 inhibitor, mineraloil, any type of alcohol, a non-ionic detergent such as Triton X-100,emulsifying wax, glycerol monostearate (GMS), isopropyl myristate (IPM),and about 60-80% water.

In yet other embodiments, a cream or lotion formulation of the inventioncomprises about 0.001% to about 1% (w/w) of rapamycin, mineral oil, anytype of alcohol, a non-ionic detergent such as Triton X-100, emulsifyingwax, glycerol monostearate (GMS), isopropyl myristate (IPM), and about60-80% water.

In yet other embodiments, an ointment formulation of the inventioncomprises about 0.001% to about 1% (w/w) of a mTORC1 inhibitor in anaqueous solution having about 10-50% (w/w) of DMSO and about 10-50%(w/w) of PEG and about 1-60% (w/w) petrolatum.

In yet other embodiments, an ointment formulation of the inventioncomprises about 0.001% to about 1% (w/w) of rapamycin in an aqueoussolution having about 10-50% (w/w) of DMSO and about 10-50% (w/w) of PEGand about 1-60% (w/w) petrolatum.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for topical administration, such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, transdermal patches, and solutions or suspensionsthat are adapted for controlled-release are encompassed by the presentinvention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds.

In certain embodiments of the invention, the compositions of theinvention are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials and Methods

Unless otherwise noted, all cell lines, starting materials, reagents andcell lines were obtained from commercial suppliers and used withoutfurther manipulation.

Cell Culture and Cell Culture Reagents

Cell culture experiments utilizing NRTIs were of the following design.Cultures of either human lung or cardiac fibroblasts were cultivatedaccording to standard culture protocols for these cells (Cristofalo, etal., Journal of Tissue Culture Methods 1980, 6:117-121). Parallel setsof cultures were maintained in normal growth media or in normal growthmedia with the addition of 1 nM rapamycin. Cultures were maintained with1 nM rapamycin (Enzo Biologicals) for two weeks before exposure toNRTIs. Cell cultures were exposed to NRTIs at indicated concentrationsin individual experiments (generally 10-20 μg/ml) for 7 days, with achange of media and fresh NRTIs at day 4. Mitochondrial measurements,bioanalyzer measurements, immunoblotting for protein expression andphosphorylation status, and assays for senescence were performed at theend of this 7-day period.

Cell culture reagents were from Cellgro, unless indicated. WI-38fetal-lung primary human fibroblasts or human cardiac fibroblasts weregrown in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine,MEM vitamins, and MEM non-essential amino acids. Cells were maintainedin a 37° C. in 5% CO₂ incubator. For treatment studies, cells weretreated with 1 nM rapamycin (Enzo Biologicals) for two weeks beforetreatment with NRTIs. Cells were maintained by trypsinization andreseeding at a cell density of 1×10⁴/cm² every 7 to 10 days.Emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) were kindlyprovided by the NIH AIDS Research & Reference Reagent program. Duringthe 7-day exposure to the NRTIs, the pharmacologic inhibitors PD98059(10 μM, Santa Cruz Biotechnologies), BI-D1870, GW5074, and SB203580 (10μM, Enzo Biologicals) were added to the culture medium either during thefinal 48 hours or for the final 2 hours depending upon the experiment.During the 7-day exposure to NRTIs, Trolox (500 μM) and N-acetylcysteine(100 μM, Acros Organics) were added every other day. Mito-Q (20 nM) andTPP (20 nM) were kindly provided by Dr. Brett Kaufmann at the Universityof Pennsylvania School of Veterinary Medicine. Vector and mt-catalaseadenoviruses (MOI's 25, 50, and 75) were purchased from Gene TransferVector Core from the University of Iowa.

Western Blotting and Co-Immunoprecipitation

Cell protein extracts were prepared by extracting withradioimmunoprecipitation assay (RIPA) buffer containing a proteaseinhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors, NaF andsodium orthovanadate. Protein concentration was quantified using abicinchoninic acid (BCA) assay (Pierce Biotechnology). Western blotanalysis was performed using 15 to 30 μg of protein extracts that wererun on SDS-PAGE and transferred onto nitrocellulose (Biorad) membranes.Blots were incubated with antibodies specific for: beta-actin(Sigma-Aldrich), TFAm, parkin, p16, phospho(S82)HSP27, HSP27 (Santa CruzBiotechnologies), p53, 21, catalase, MDM2 (EMD Millipore), VDAC,phospho(S235/236)-ribosomal protein S6, ribosomal protein S6,phospho(S166)-MDM2, phospho(S473)-AKT, AKT, beta-tubulin (CellSignaling), p62 (Enzo Biologicals), IL-6 (NeoBiolab), lamin B1, PINK 1,and Mitoprofile Total OXPHOS Cocktail (complex I-NDUFB8 subunit,CII-SDHB subunit, CIIIUQCRC2 subunit, and CIV-mitochondrial COX1subunit) (Abcam) according to manufacturers' instructions. Western blotswere visualized using IRDye 680 and 800 LI-COR secondary antibodies on aLI-COR Odyssey using LI-COR Odyssey software version 3.0 (LI-CORBiosciences). For co-immunoprecipitation, cell protein extracts wereprepared in HNTG buffer containing a protease inhibitor cocktail andquantified using BCA assay. Protein extracts at the concentration of 0.5mg were pre-cleared with protein A/G beads (Santa Cruz Biologicals) for30 minutes. After centrifugation at 2,000 rpm for 5 minutes at 4° C. thesupernatant was incubated with the primary antibody at manufacturer'srecommended concentration overnight at 4° C. An IgG antibody (CellSignaling) was used as a non-specific control. Immunocomplexes wereprecipitated using 100 μl of protein A/G beads overnight at 4° C.Immunocomplexes bound to the beads were centrifuged at 2000 rpm for 5minutes at 4° C. and washed three times using HNTG buffer. Following theaddition of 2× sample buffer and boiling at 100° C., protein sampleswere used for western blotting.

Total ROS, Mitochondrial Membrane Potential, Mitochondrial Mass, andSuperoxide Anion Assessments

Mitochondrial membrane potential was detected by incubating cells with25 nM TMRE (Molecular Probes). Mitochondrial mass was evaluated byincubating the cells with 100 nM Mitotracker Green FM (MolecularProbes). Mitochondrial superoxide anion levels were detected byincubating the cells with 5 μM MitoSox Red (Molecular Probes). Totalcellular levels of ROS were detected by incubating the cells with 10 μM2′,7′-dichlorofluoroscein diacetate (DCF-DA; Sigma-Aldrich) in 1% fetalbovine serum supplemented MEM and washing twice with Krebs Ringerphosphate glucose buffer (145 mM NaCl, 5.7 mM NaH₂PO₄, 4.86 mM KCL, 0.54mM CaCl₂, 1.22 mM MgSO₄, and 5.5 mM glucose) following the incubationperiod. For the aforementioned assessments, incubation was performed at37° C. in 5% CO₂ for 30 minutes and cells were harvested in 2.5%trypsin-EDTA with serum-containing medium. Cells were immediatelyanalyzed with a Guava EasyCyte Mini employing the Guava Express Plusprogram (Guava Technologies).

Mitochondrial Respiration Measurements

Mitochondrial function was measured using the XF cell mito stress testkit on a Seahorse XF24 Bioanalyzer (Seahorse Bioscience). Cells wereseeded at a density of 15,000 cells per well in an XF24 microplate.Following acquisition, results were normalized based upon 10⁶ cellscounted using a Guava EasyCyte Mini (Millipore), where applicable.Plates were loaded into the Bioanalyzer that had been pre-loaded withthe sensor cartridge containing oligomycin, carbonyl cyanidep-triflouromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A.Oxygen consumption was measured in triplicate prior to and followingsequential addition of oligomycin, FCCP, and rotenone/antimycin A.Respiration rates and proton leak were assessed as outlined in publishedmethods (Hill, et al., Biological chemistry 2012, 393:1485-1512).Mitochondrial respiration was calculated based upon oxygen consumptionrate measurements in triplicates from cells seeded in at leastquadruplicates. Basal respiration represents the initial oxygenconsumption rate measurements and maximal respiration represents theoxygen consumption rate measurements following FCCP addition. ATP-linkedrespiration is represented as the oligomycin-sensitive oxygen change tobasal oxygen consumption rate. Proton leak represents the oligmycininsensitive oxygen consumption rate. Non-mitochondrial sources of oxygenconsumption were subtracted by normalizing to the rotenone/antimycinA-insensitive oxygen consumption rate measurements. All data wasnormalized to cell number by counting cells in each well at thecompletion of the mitochondrial assessments.

Senescence Associated Beta Galactosidase Detection

Assessment of SA-β-gal activity was performed by plating cells followingrespective treatments at low density (0.5×10⁴/cm²) to prevent falsepositive staining known to occur in high density cultures. Seeding wasperformed for SA-β-gal staining following NRTI treatment for one weekand staining was performed 24 hours following seeding. Cells were washedwith PBS, fixed with 2% formaldehyde-0.2% glutaraldehyde for 5 minutesat room temperature, washed with PBS once more, and incubated overnightat 37° C. in a staining solution containing 50 mg/ml X-gal, 100 mMpotassium ferricyanide, 100 mM potassium ferrocyanide, 5 M NaCl, 1 MMgCl₂, and 0.2 M citric acid/phosphate buffer (pH 6.0). Followingincubation, cells were washed three times with PBS and at least 500cells were counted for each sample in triplicates. Positive cells (bluecells) were expressed as a percentage of total cells.

Immunofluorescence

Cells were seeded onto acid-washed coverslips at standard densityfollowing the designated treatment. Following 24 hours, cells were fixedusing 4% paraformaldehyde, permeabilized using 0.1% Triton-PBS, andblocked with applicable animal serum. Immunofluorescence was performedto visualize the mitochondria using a cytochrome C antibody (EMDMillipore) and co-stained with a FRAP antibody (Santa Cruz).Mitochondria were also visualized using lentiviral transduction of amitochondrial tagged GFP (Vector Core, University of Pittsburgh) andselected using puromycin. Following applicable experimental treatment,immunofluorescence was performed on mitochondrial-expressing cells usinga Raptor antibody (Bethyl Laboratories). Following primary antibodyincubation, staining was performed using Alexa-Flour Secondaries (LICORBiosciences) and DAPI as a nuclear stain (10 ng/ml) and preserved withVectaShield mounting media (Fisher Scientific). Cells were imaged usingdeconvolution or confocal microscopy for co-localization eventquantification.

Statistical Analysis

Results are representative of at least three independent experiments andstatistical significance was determined using an unpaired two-tailedStudent's t-test. Data sets were subjected to normality tests to verifynormal distribution of data. One-way Anova with Bonferroni post-hocanalysis was performed on multiple comparison groups involving control,NRTI, rapamycin, and rapamycin NRTI where appropriate.

Formulations

An exemplary non-limiting emulsion formulation of the invention(referred to as “Formulation R” hereinafter) comprises about 0.001% toabout 1% (w/w) of rapamycin, palmitate at about 4-6%, glycerin at about6-8%, and the balance consisting of water.

Administration to Patients

Patients presenting to a physician with a diagnosis of dermal atrophy,seborrheic keratosis, actinic keratosis, lentigines, senile pseudoscars,or coarse wrinkles were selected for administration of a composition ofthe invention. Dermal thickness was quantified using a Mitoutoyo digitalcaliper with certified accuracy to 0.001 mm. Lesions (actinic keratosis,psuedoscars, coarse wrinkles, were evaluated utilizing the Investigatorevaluation rating scale for severity. Formulation R was provided to thepatient with instruction for administration 1-2 times per day for aninitial 2-week period. Patients were advised to cease application ofFormulation R at any sign of adverse reaction in the area ofapplication. Following the 2-week application period, dermal thicknessand lesion severity was monitored on a weekly basis.

Example 1: Mitochondrial Effects of Nucleoside/Nucleotide Analogs areRelieved by Rapamycin

The effects of combination treatment with TDF and FTC (referred to asNRTIs for simplicity) were examined at concentrations relevant to serumlevels in patients receiving anti-retroviral therapy, on mitochondria inboth human cardiac and lung fibroblasts. Parallel cultures were grown inthe additional presence of 1 nM rapamycin. This concentration ofrapamycin was found to extend replicative lifespan and improve themitochondrial profile of human fibroblasts. Exposure to NRTIs for 7 daysproduced a significant increase in mitochondrial membrane potential,mitochondrial ROS production, and mitochondrial mass in the humancardiac fibroblasts (FIGS. 1A-1C). Similar results were observed in thehuman lung fibroblasts. Additionally, total cellular ROS increasedsignificantly following exposure to NRTIs in both fibroblast populations(FIG. 1D). Cultures grown in the presence of rapamycin did not exhibitthe same level of increase in mitochondrial membrane potential,mitochondrial ROS, or total cellular ROS following exposure to NRTIs.

An indirect assessment of mitochondrial activity was performed using aSeahorse Bioanalyzer using cultures maintained in standard culturemedium or maintained in the presence of rapamycin. The calculated ratesof basal respiration, maximal respiration, ATP-linked respiration, andproton leak increased significantly when human cardiac fibroblasts wereexposed to NRTIs (FIGS. 1E-1I). Rapamycin-treated cells hadsignificantly lower basal respiration and did not exhibit an increase inbasal or maximal respiration when exposed to NRTIs (FIGS. 1F-1G).ATP-linked respiration was increased by NRTI exposure and was reduced inrapamycin-treated cultures (FIG. 1H). In addition, the rapamycin-treatedcells did not exhibit an increase in proton leak following exposure toNRTIs (FIG. 11I). Human lung fibroblasts gave similar results whensubjected to the same analyses (i.e., basal and maximal respirationincreased significantly, as did proton leak in cells treated withNRTIs). Similar to the cardiac fibroblasts, rapamycin-treated lungfibroblasts exhibited no increase in these parameters following exposureto NRTIs.

Example 2: Alterations in Electron Transport Chain Components inResponse to Nucleoside/Nucleotide Analogs and Rapamycin

The effect of NRTI exposure on steady state levels of a subset ofelectron transport chain proteins was examined. Levels of NADHdehydrogenase (ubiquinone) 1 beta subcomplex, 8 (NDUFB8) of complex I,succinate dehydrogenase (ubiquinone) ironsulfur subunit (SDHB) ofcomplex II, ubiquinol-cytochrome c reductase core protein II (UQCRC2) ofcomplex III, and cytochrome c oxidase subunit I (mt-CO1) of complex IVall increased following exposure to NRTIs (FIG. 2A). Similarly, thesteady state level of the outer membrane voltage dependent channel(VDAC) increased in cells exposed to NRTIs while in contrast, steadystate levels of the ATP synthase alpha subunit 1 (ATP5A) were unchanged(FIG. 2A).

Cells treated with rapamycin expressed lower steady state levels ofNDUFB8, SDHB, UQCRC2, and mt-CO1, while levels of ATP5A were similar tocontrol cells. In addition, ETC protein levels were not elevated whenrapamycin-treated cells were exposed to NRTIs (FIG. 2A).

Levels of proteins involved in mitochondrial clearance, Pink 1 andParkin, were also examined (FIG. 2B). Exposure to NRTIs caused anincrease in Pink and the cleaved form of Pink 1, as well as a decreasein Parkin. The level of the autophagy cargo loading protein p62/SQSTM1increased in response to NRTIs, but decreased in response to rapamycin(FIG. 2C).

Differences in mitochondria-related gene expression was examined using ananostring approach which allows multiplex evaluation of mRNA species inthe absence of amplification and provides a direct count of mRNAmolecules. The mRNA levels paralleled the changes observed in proteinlevels as mRNA levels for all subunits increased in cells exposed toNRTIs while rapamycin decreased mRNA levels for all subunits (FIGS.2D-2H).

Example 3: Senescence Response to Mitochondrial Dysfunction andProtection by Rapamycin

Molecular markers of the senescence program were examined followingexposure to NRTIs (FIGS. 3A-3F). Levels of p53, p21, and p16 increasedin fibroblasts exposed to NRTIs, while in rapamycin treated cells thelevels of these senescence associated proteins did not increase (FIG.3A). In addition, levels of lamin B1, which is known to decrease duringsenescence, decreased in cells exposed to NRTIs. Rapamycin preventedthis decrease (FIG. 3A). Additionally, intracellular levels of IL-6,which is a component of the senescence associated secretory program,increased following exposure to NRTIs. Consistent with a block in thesenescence program, rapamycin treated cells showed no increase in thelevels of IL-6 following exposure to NRTIs (FIG. 3A). The percentage ofthe cell population expressing the senescence marker, senescenceassociated β-galactosidase (SA-β-gal) following exposure to NRTIs wasexamined. A dose dependent increase in the percent of cardiacfibroblasts expressing SA-β-gal was observed (9% in control cellscompared to 28% and 48% in cells exposed to 10 or 20 ug/ml of NRITsrespectively, FIG. 3B). In contrast, rapamycin treated cultures showedno increase in SA-β-gal (FIG. 3B). Analysis of mRNA levels for p21 andlamin B1 by nanostring revealed that steady state mRNA levels varied inparallel with protein levels, p21 mRNA significantly increased inresponse to NRTIs while lamin B1 mRNA levels significantly decreased(FIGS. 3C-3D), which was similar to the effect on the expression of p21and lamin B1 during replicative senescence (FIGS. 3E-3F).

Lung fibroblasts showed identical changes in response to NRTIs in termsof senescence markers and the protection afforded by rapamycintreatment. There was no reduction in viability of cells exposed to NRTIsas judged by vital dye exclusion assay under any of the conditionstested and no apparent markers of apoptosis, such as caspase cleavage.Alkaline comet assay showed no evidence of increased DNA damage,indicating that the response to NRTIs was primarily a growth inhibitionand not due to DNA damage. The interaction between p53 and its keyregulator. MDM2, was also examined following NRTI exposure. Exposure toNRTIs reduced the association between p53 and MDM2 and increased MDM2phosphorylation at serine 166. In addition, the use of a proteasomeinhibitor revealed high molecular weight forms of p53 that wereprominent in rapamycin-treated cultures but reduced following NRTIexposure.

The role of mitochondrial ROS in the NRTI-induced senescence responsewas examined by treating control cultures (grown without rapamycin) withmito-Q, a ROS scavenger that targets mitochondrial ROS or by introducinga mitochondrial targeted catalase (mt-catalase) into cardiacfibroblasts. Mito-Q ameliorated both mitochondrial ROS productionfollowing exposure to NRTIs and the increase in senescence-associatedproteins p53, p21, and p16 (FIG. 4A). In addition, the percentage ofcells positive for SA-β-gal staining was reduced in cells treated withmito-Q (FIG. 4B). Similar results were obtained when cells were infectedwith the mt-catalase construct. The increase in senescence-associatedproteins p16 and p21 following NRTI exposure was prevented and thepercentage of cells staining positive for SA-β-gal was significantlyreduced (FIGS. 4C-4D). The treatment of cells exposed to NRTIs with acombination of the antioxidants trolox and N-acetylcysteine (trolox/NAC)also prevented the induction of senescence. Markers of senescence werereduced including SA-β-gal expression and levels of p16 and p21, whilethe levels of lamin B1 were maintained, consistent with an inhibition ofthe senescence program (FIGS. 4E-4F).

Example 4: Activation of mTORC1/p70S6K Signaling in Senescence

The possibility that MDM2 phosphorylation, mediated by p70S6K, is acomponent of the cellular response to NRTIs was investigated. It wasfirst verified that exposure to NRTIs increased both p70S6K activity (byexamining the phosphorylation status of the ribosomal S6 protein) andphosphorylation status of MDM2. Phosphorylation of both the ribosomal S6protein and MDM2 increased in cardiac fibroblasts following exposure toNRTIs (FIG. 5A). Consistent with the inhibition of senescence,rapamycin-treated cultures showed a complete lack of ribosomal S6phosphorylation and no increase in MDM2 phosphorylation (FIG. 5A). Therole of p70S6K in MDM2 phosphorylation in this setting was examinedusing the specific p70S6K inhibitor PF-4708671. The phosphorylation ofMDM2 in response to NRTIs was inhibited by PF-4708671, as wasphosphorylation of the ribosomal S6 protein, which served as a positivecontrol for the inhibitor (FIG. 5B).

The impact of inhibitors of MAPK on ribosomal S6 and MDM2phosphorylation in cells exposed to NRTIs was also examined. Cellsexposed to NRTIs were treated with inhibitors of 3 members of the MAPKsignaling pathway, MEK1 (U0126), Raf1 (GW5047), and p90RSK (BI-D1870).All 3 MAPK inhibitors caused some decrease in ribosomal S6phosphorylation, but the p90RSK inhibitor, BI-D1870, had the greatestimpact (FIG. 5C). In contrast, an inhibitor of the p38 stress activatedkinase, SB-203580, had no effect on the phosphorylation of ribosomalprotein S6 or MDM2 (FIG. 5D). The effects of rapamycin, the p70 S6kinase inhibitor, the inhibitor of p90RSK, and Trolox/NAC on ribosomalS6 and MDM2 phosphorylation events in response to NRTIs were verified inhuman lung fibroblasts.

The dependence of NRTI-induced p70S6K activity on ROS was examined bytreating cells exposed to NRTIs with trolox/N-acetylcysteine andexamining ribosomal S6 phosphorylation. Both ribosomal S6phosphorylation and MDM2 phosphorylation were decreased when cellsexposed to NRTIs were treated with trolox/N-acetylcysteine (FIG. 5C).Involvement of the NADPH oxidase system was examined using apocyanin, aninhibitor of NADPH oxidases. However, treatment of NRTI-exposed cellswith apocyanin did not prevent the increase in ribosomal S6phosphorylation. Similar results in terms of p7-S6 kinase and MDMphosphorylation were obtained when human lung fibroblasts were exposedto NRTIs.

In order to examine the potential role of mitochondrial ROS in theactivation of mTOR activity, cells were treated with either mito-Qduring NRTI exposure or the mt-catalase adenovirus prior to exposure toNRTIs. Both of these interventions, mito-Q and expression of themt-catalase, reduced ribosomal S6 phosphorylation and MDM2phosphorylation following exposure to NRTIs (FIGS. 6A-6B). Additionally,mito-Q treated cells were examined by Seahorse Bioanalyzer to assessmitochondrial activity. This analysis revealed that mito-Q treatmentpartially alleviated the increase in basal respiration, ATP-linkedrespiration, and proton leak, while maximal respiration was lessaffected (FIGS. 6C-6F). In addition, mito-Q treated cells exhibited alower level of mitochondrial ROS when exposed to NRTIs (FIG. 6G).

To determine whether increased ROS can directly induce phosphorylationof the ribosomal S6 protein, cardiac fibroblasts were placed inserum-free medium for 48 hours to abrogate growth factor signaling thatmight influence mTORC1 activity. The cells were then exposed to hydrogenperoxide at concentrations ranging from 1 to 400 μM for 2 hours. Bothribosomal S6 phosphorylation and MDM2 phosphorylation were increased atthe lower concentrations of hydrogen peroxide, with maximal activationat 50 μM and inhibition of both ribosomal S6 phosphorylation and MDM2phosphorylation occurred at concentrations above 100 μM (FIG. 7A). Theresponse to hydrogen peroxide differed from the serum response which,consistent with a proliferative response, led to phosphorylation of theribosomal S6 protein, but not MDM2 (FIG. 7A, far right). To determinewhether an induction of mitochondrial ROS can lead to activation of mTORsignaling, cell were exposed to a range of concentrations of rotenoneand the phosphorylation of the ribosomal S6 protein was examined.Exposure of cells to nanomolar concentrations of rotenone lead toincreased S6 phosphorylation with a sharp inhibition of S6phosphorylation occurring at 50 nM (FIG. 7B).

Example 5: Association of mTORC1 with Mitochondria in Response toMitochondrial Stress

The possibility that mitochondrial association with the mTORC1 complexis enhanced when cells are exposed to NRTIs was examined using humancardiac fibroblasts expressing a green fluorescent protein fused to amitochondrial-targeting sequence (mt-GFP). These cells were exposed toNRTIs and fixed for immunofluorescence using antibodies that recognizethe mTORC1-specific component. Raptor. In cells exposed to NRTIs, Raptorwas associated with mitochondria to a greater degree than in untreatedcells (FIGS. 8A-8C).

The role of mitochondrial ROS in senescence was examined by treatingsenescent cardiac fibroblasts with the mitochondrial ROS scavengermito-Q or by introducing a mitochondrial targeted catalase. Initially,levels of mitochondrial and total cellular ROS were examined in latepassage cells. This assessment revealed elevated levels of mitochondrialROS and total cellular ROS in late passage cells (FIGS. 9A-9B).Assessment of mitochondrial activity by Seahorse Bioanalyzer revealedelevated respiration, consistent with previous studies in senescentcells using isolated mitochondria. Bioanalyzer analysis indicated thatboth basal and ATP-linked respiration rates were significantly increasedin senescent cells compared to early passage cells, as was proton leak(FIGS. 9C-9G).

The progressive increase in ribosomal S6 and MDM2 phosphorylation duringreplicative senescence was verified in cardiac fibroblasts (FIG. 9H). Inorder to test the dependence of phosphorylation of the ribosomal S6protein in senescent cells on ROS, senescent cells were treated with theROS scavengers trolox and N-acetylcysteine. In parallel, the mt-catalasewas introduced into senescent cells to reduce mitochondrial ROSproduction. Both of these interventions reduced the high basal levels ofribosomal S6 phosphorylation typical of senescent fibroblasts andreduced levels of phosphorylated MDM2. In addition, treatment with thep90RSK inhibitor, BI-D1870, also reduced both ribosomal S6 and MDM2phosphorylation in senescent cells (FIG. 9I).

Example 6: In Vivo Topical Application of Rapamycin

A single site open label study was performed. A patient presenting withan area of dermal atrophy and actinic keratosis on the hand wasevaluated for application of Formulation R, the contralateral hand withsimilar dermal thickness but no actinic keratosis was used as a control.The patient was provided with Formulation R with instructions for twicedaily application.

Following a 2-week (14 day) period, both actinic keratosis and dermalthickening showed signs of improvement as self-reported by the patientand found by study personnel. Evaluation of dermal thickness revealed anincrease in dermal thickness of ˜20% (1.6 SD 0.13 untreated versus 1.9SD 0.19 treated). Actinic keratosis was improved from a rating of 3 to arating of 2. No evidence of adverse reaction was observed or reported bythe patient at 14 day follow-up visit. Continued application ofFormulation R beyond the initial 14 day period provided continuedbenefit while administration of the carrier formulation withoutrapamycin has no influence on dermal thickness in the contralateralhand.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. A method of treating an age-related dermal disorderin a mammalian subject, the method comprising topically administering tothe subject a composition consisting essentially of about 0.001-0.01%(w/w) of a mTORC1 inhibitor wherein the mTORC1 inhibitor is rapamycin,or a salt, solvate, enantiomer, or diastereoisomer thereof, wherein theproliferative potential of dermal cells is maintained; and wherein theage-related dermal disorder is at least one selected from the groupconsisting of age-related dermal atrophy, seborrheic or actinickeratosis, pseudoscars, lentigines, focal areas of dermal thickening,and coarse wrinkles.
 2. The method of claim 1, wherein the subject is ahuman.
 3. The method of claim 1, wherein the composition furthercomprises a dermatologically acceptable carrier.
 4. The method of claim3, wherein the dermatologically acceptable carrier is at least oneselected from the group consisting of a solvent, lubricant, emollient,emulsifier, moisturizer, thickening wax, softener, fragrance,preservative, and artificial color.
 5. The method of claim 3, whereinthe dermatologically acceptable carrier comprises petrolatum.